KR20220005446A - Liposome production method - Google Patents

Abstract

The present invention relates to a method for the production of liposomes in order to obtain a high encapsulation efficiency of an encapsulated formulation, excluding the reduced number of production steps, i.e., the extrusion steps of conventional liposome production processes. The liposomes of the present invention are intended to deliver therapeutic agents such as anticancer agents, antioxidants, anti-inflammatory agents, antipyretics, antibiotics, antiviral agents, antirheumatic agents, analgesics, growth-factors, or mixtures thereof.

Description

Liposome production method

The present invention relates to a method for the production of liposomes for obtaining high encapsulation efficiency of encapsulants with fewer production steps.

Liposomes are defined as artificial microscopic vesicles composed of an aqueous core surrounded by one or more concentric phospholipid layers (lamellae) [1]. Because liposomes are non-toxic and biodegradable because they are composed of naturally occurring substances, they are receiving much attention as carriers for various therapeutic agents [2]. Liposomes show a wide range of potential applications because they can contain hydrophilic (in the aqueous compartment), hydrophobic (in the lipid membrane), and amphiphilic substances (in the lipid aqueous interface) [3]. In addition, the biologically active substances encapsulated in the liposomes are protected from immediate dilution or degradation. For all these reasons, liposomes are the most widely used nanocarrier systems since their discovery.

The widespread use of liposomes for multiple purposes has created the need to develop an efficient and reproducible preparation method in the simplest possible way. There are various modifications to the method for preparing liposomes. Because of its simplicity, most laboratories use lipid thin-film hydration, first described in 1965 [4]. However, the film method tends not to be suitable for large-scale production. In addition, there is a concern about the use of a chlorine-based solvent.

The ethanol injection method is an interesting technique for GMP scale-up liposome production. It offers several advantages: simplicity, GMP-friendly solvent, fast implementation and reproducibility, the fact that it does not cause lipolysis or oxidative modification. The ethanol injection method was first reported in 1973 by Batzri and Korn [5] as one of the first alternatives to fabricate small unilamellar vesicles (SUVs) without sonication. By immediately diluting the ethanol in the aqueous phase, the lipid molecules precipitate to form bilayer planar fragments. Through energy dissipation in the system (by agitation and/or sonication), fragments of these lipid bilayers tend to reduce the exposure of the hydrophobic portion of the molecule to an aqueous environment, resulting in such a quasi-spherical structure. The curvature of the fragment occurs. In the next few years, several studies have shown that the manufacturing parameters of the ethanol injection technique (lipid concentration and composition, infusion rate, temperature of both phases) for the properties of the generated liposomes (size distribution, zeta potential, drug encapsulation efficiency, etc.) , stirring speed, etc.) were investigated [6].

In conventional ethanol injection methods, the ethanol phase is a small proportion compared to the aqueous phase, typically 5-10%. After evaporation of the ethanol, the liposome dispersion is extruded to reduce the vesicle size. Briefly, the conventional ethanol injection method consists of three main steps:

1. Lipid (ethanol) injection into the aqueous phase;

2. Extrusion to reduce liposome size; and

3. Eliminate unencapsulated formulations.

In general, liposomal therapeutic or contrast agent loading is accomplished by passive or active methods:

Passive loading involves dissolution of the dried lipid film in an aqueous solution containing the agent of interest. This approach can only be used with water-soluble formulations, and loading efficiencies are often low (less than 5%). This method can be used for a wide range of compounds irrespective of their chemical structure.

Active loading involves the internalization of an agent driven by a liposomal transmembrane pH gradient [7]. This process can be very efficient (>95%), with high intraliposome concentrations and minimal waste of expensive chemotherapeutic agents.

However, this method (active loading) requires that molecules have different protonation states at the extreme pH of the buffer used inside and outside the liposome. A molecule given in this way will diffuse the lipid bilayer when two different pHs are established inside and outside the liposome. Thus, the pH gradient is the driving force for transport and retention of amphiphilic weak bases and acids [8].

Active loading approaches for weakly basic amine therapeutics or imaging agents using a transmembrane ammonium sulfate gradient have also been reported in the literature. In this case, the ammonia gradient induces a pH gradient, which actively transports the agent into the liposome. The sulfate then acts as a counter ion to the ionized agent, allowing it to precipitate within the liposome. This method was applied to the production of liposomal doxorubicin on Doxil. Myocet is another example of a remotely loaded liposomal doxorubicin, although the pH gradient is established with citric acid [9].

In the active loading process, after the liposome is prepared by the conventional ethanol injection method, the liposome-extra-phase is removed, then the agent is added to the liposome-extra-phase and the liposome is incubated to proceed with the remote loading process. Briefly, the active loading process consists of five main steps:

1. Lipid (ethanol) injection into the aqueous phase;

2. Extrusion to reduce liposome size;

3. Removal of liposome-extraphase;

4. Incubation of formulations with liposomes; and

5. Remove the unencapsulated formulation.

WO/2013/084208 (Paulo A. et al ., 2013, Liposomes and its production method) describes a lipid membrane hydration method for producing liposomes.

US4752425A (Martin F. et al ., 1988, High-encapsulation liposome processing method) describes a method for producing liposomes using chloroform. The resulting liposomes are larger than 1.5 microns and require extrusion for size reduction (if the original encapsulated formulation is lost).

US5549910A (Szoka F. et al ., 1994, Preparation of liposome and lipid complex compositions) describes a method for obtaining liposomes containing compounds exhibiting low solubility in water, alcohol, and halogenated hydrocarbon solvents. In this method, the lipid is dissolved in a solution of an aprotic solvent and may further contain a lower alkanol if necessary to dissolve it. This method requires extrusion to obtain liposomes of defined size.

US20120171280A1 (Zhang Y, 2011, Method of making liposomes, liposome compositions made by the methods, and methods of using the same), an aqueous solution comprising ethylenediaminetetraacetic acid (EDTA) for encapsulating ascorbic acid or a salt thereof, Methods for obtaining liposomes are described. The liposome composition has a selected average particle size diameter of about 200-500 nm.

US 5316771A (Barenholz, Y. et al ., 1994, Method of amphiphatic drug loading in liposomes by ammonium ion gradient) describes the active loading of weakly amphiphilic drugs into liposomes using a transmembrane gradient.

US 5939096A (Clerc, S.Y. and Barenholz, 1999, Stably encapsulating a weak acid drug in liposomes, at a high concentration) describes liposomes encapsulated with a weakly acidic drug in high concentration. This method used a proton shuttle mechanism involving salts of weak acids to create a higher internal/lower external pH gradient.

WO201364911 relates to methods and compositions for producing lipid-encapsulated negative-charge therapeutic polymers such as nucleic acids, proteins, and peptides encapsulated in a lipid layer.

WO0105374 relates to methods and compositions for producing lipid-encapsulated charged therapeutic agent particles after mixing of a preformed lipid vesicle, a charged therapeutic agent (having an opposite charge to the lipid) and a destabilizing agent.

general content of the invention

The method herein has the advantage of achieving the same or better small molecule encapsulation efficiency in the target liposome than the previous method without additional processing steps to generate nanoparticles. Multicharged molecules that are negatively charged in their structure at neutral pH (5-8), such as methotrexate and doxorubicin, are better encapsulated in this way. Methotextrate is doxorubicin with high encapsulation rate and reduced number of steps (Tables 2 and 3)

In the proposed alternative method, a higher encapsulation efficiency of the therapeutic or imaging agent is achieved using a pre-concentration method with an ethanol:aqueous phase in a similar volume ratio, and the liposomes can be diluted last. The novel proposed method provides a desirable reduced number of steps (only two) in industrial processes. These features were aggregated for the first time in the same way, which differentiates them from those reported in the literature.

The widespread use of liposomes for multiple purposes has created a need to develop manufacturing methods that are efficient, reproducible and should be the simplest possible. Existing methods remain difficult for industrial scale-up and/or to achieve low encapsulation efficiencies of agents of interest. As such, there is an urgent need to develop a method that can reduce the number of steps and achieve high encapsulation efficiency of the encapsulant.

The lipids used to generate liposomes can be altered or modified to customize the properties of the liposome surface and membrane layer. There are different types of lipids depending on the charge, such as neutral, cationic, and anionic. Addition of organic molecules to the phosphate head group yields various phospholipid species such as phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylcholine (PC). All these lipids can be used for liposome production.

Unmodified liposomes do not survive long in circulation because they are removed by macrophages. One of the first attempts to overcome this problem, including, for example, steroids, focused on manipulating lipid membrane components to modulate bilayer fluidity. In this way, the liposome formulation of the present invention may preferentially contain cholesterol (CH) which may vary in a molar ratio of 35-55%, preferably 35-40%. Incorporation of cholesterol into liposomes has been shown to increase the packing of phospholipids in the lipid bilayer, thereby reducing their interaction with blood proteins.

In addition, polyethylene glycol (PEG), a synthetic polymer, was preferentially incorporated into the liposome. PEG-containing liposomes had less binding to blood proteins and decreased RES uptake, prolonging the duration of liposomes in circulation. This extended the blood circulation of conventional liposomes to drug delivery, and the conjugated phospholipid DSPE-MPEG was incorporated into the lipid film of this novel formulation in a molar ratio that could vary between 4-12%, preferably 5-10%.

It has also been demonstrated that surface-modified liposomes with gangliosides have extended circulation times in the bloodstream compared to non-modified liposomes. This property is potentially useful for the application of gangliosides in immunotherapy. Several glycolipids were tested in a study of RES uptake of liposomes after intravenous injection: the glycolipid GM1 (brain-tissue-derived monosialoganglioside) significantly reduced RES uptake when incorporated into the liposome surface, and the formulation was put into circulation for several hours. remained

Active targeting uses specific modification of the liposome surface with a targeting ligand, so that it can accumulate at a target site or be delivered intracellularly to a target cell. Incorporation of a specific ligand into the liposome allows its contents to be released into the cell by receptor-mediated endocytosis. Targeting agent incorporation at the membrane surface can be achieved either by conjugation to phospholipids or fatty acyl chains or by incorporation into the lipid membrane.

There are various methods for the preparation of liposomes, and there are various modifications. In the ethanol injection method (5% ethanol), a portion of the aqueous solution is passively encapsulated inside the vesicle together with the water-soluble material. Although the advantage of this method is its simplicity, only a very small proportion of the water-soluble therapeutic agent or imaging agent can be encapsulated in this way.

In remote loading, empty liposomes are usually prepared in initial salt or low pH buffer. The liposome-traumatic phase is then removed using dialysis or size exclusion chromatography or by titrating the pH to slightly basic conditions. Finally, the agent is added to the extra-liposome step and the liposomes are cultured to proceed with the remote loading process [6]. The number of steps involved makes the production process difficult to scale and constitutes a barrier to further development of this standard approach.

Therefore, the focus is on optimization of agent encapsulation inside liposomes with reduced production steps. In practice, only a small amount of the formulation used is encapsulated (eg 3-5% for methotrexate drug), a large amount of formulation is wasted and this can be a problem in the scale-up process. Numerous conditions were tested at different stages of the production process to increase the encapsulated formulation. That is, in the aqueous phase of the organic phase containing phospholipids (as opposed to instead), without removal of ethanol, and at room temperature instead of 70ºC, injections are performed at different injection rates and different concentrations of the organic phase (from 5% to 95%).

The present invention consists of a novel method for preparing liposomes in which a hydrophobic component of liposomes is dissolved in ethanol and injected into an aqueous phase at a rate of about 2-4 ml/min under vigorous stirring. The initial volume ratio of ethanol:aqueous phase is 1/1. After ethanol evaporation or tangential flow filtration, the liposome dispersion should be diluted 1 to 10 times to the desired final concentration.

In one embodiment of the present invention, the pre-concentration method comprises two main steps:

· After injection of lipids (ethanol) into the aqueous phase (1:1 v/v), dilute;

· Remove unencapsulated preparations.

In one embodiment of the present disclosure, this method allows to achieve high encapsulation efficiencies (eg -40%) for multicharged agents such as methotrexate with a reduced number of steps (only two). Initial pre-concentration (using a lower aqueous volume) increases the phospholipid concentration and consequently allows for higher encapsulation efficiency. In addition, using an initial 1:1 ethanol:aqueous phase volume ratio can increase the encapsulation of the formulation by maintaining a balance between the two phases with different polarities.

In one embodiment of the present specification, the present invention relates to a method for encapsulating an active ingredient in liposomes comprising the following sequential steps:

- preparing an ethanol phase by mixing phospholipids and hydrophobic molecules of a steroid (preferably cholesterol) with ethanol;

- preparing an aqueous phase with active ingredient and targeting agent in buffer solution;

- obtaining liposomes by injecting said ethanol phase into said aqueous phase at a temperature of from about 50 °C to about 80 °C, wherein the ethanol/aqueous phase volume ratio is from 1:1 to 3:2;

- removal of ethanol by evaporation or tangential flow filtration;

- removing the remaining free active ingredient in an appropriate manner, ie by tangential flow filtration;

wherein the targeting agent is a peptide conjugated to a liposome component or incorporated into a lipid membrane.

In another embodiment of the present specification, the present invention relates to a method further comprising the step of diluting the liposome dispersion by 1 to 10-fold in the further diluted aqueous phase.

In a further embodiment herein, the present invention relates to a method in which the ethanol phase is injected at a rate of approximately 2-4 ml/min.

In a further embodiment of the present specification, the present invention relates to a method in which the pouring step is carried out under stirring.

In a further embodiment of the present specification, the present invention provides that the active ingredient is a drug, in particular an anti-cancer agent, an anti-rheumatic agent, an anti-neurodegenous disease drug, an antioxidant, an anti-inflammatory agent, a drug antipyretic agent, an antibiotic, an antiviral agent, an analgesic agent, or a combination thereof. It's about how to be.

In another embodiment of the present specification, the present invention relates to a method wherein the targeting agent is a peptide selected from the following list having a degree of identity of at least 90% of the sequence: SEQ-ID. NO 1, SEQ-ID. NO 2, SEQ-ID. NO 3, or mixtures thereof; SEQ-ID. NO 1, SEQ-ID. NO 2, SEQ-ID. NO 3 , or a mixture thereof, comprises a sequence that is at least 95% identical, preferably or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical.

Methods for aligning sequences for comparison are well known in the art and include GAP, BESTFIT, BLAST, FASTA, and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find a global (over total sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. . The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI). The overall percentages of similarity and identity are methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10; 4:29. MatGAT: applications that use protein or DNA sequences to generate similarity/identity matrices). You may decide to use one of them. As will be apparent to those skilled in the art, some manual editing may be performed to optimize alignment between preserved motifs. Sequence identity values expressed as percentages in this subject were determined over the entire amino acid sequence using BLAST with basic parameters.

In another embodiment of the present specification, the present invention relates to a process wherein the ethanol concentration to the initial aqueous volume is between 40% and 60%, preferably 50%.

In another embodiment of the present disclosure, the present invention relates to a method wherein the temperature is 60°C or 70°C.

In another embodiment of the present disclosure, the present invention relates to a method wherein the active ingredient is a multicharged molecule containing at least one negative charge, in particular methottexrate or doxorubicin, at a pH of about 4 to about 7.

In a further embodiment herein, the present invention relates to a method wherein the aqueous phase is phosphate buffered saline, PBS.

In another embodiment of the present specification, the present invention relates to a method wherein the ethanol phase comprises anionic, neutral, or cationic phospholipids.

In a further embodiment of the present specification, the present invention relates to ethanol phase phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and/or derivatives thereof or mixtures thereof, in particular 1,2-dioleoyl-sn-glycero-3-phospho It relates to a method comprising polyethanolamine.

In another embodiment of the present disclosure, the present invention is directed to a method wherein the ethanol phase comprises a steroid, a stealth agent, a targeting agent, or a mixture thereof.

In a further embodiment of the present specification, the present invention relates to a method wherein the steroid is cholesterol, and/or a derivative thereof, in particular cholesteryl hemisuccinate.

In another embodiment of the present disclosure, the present invention relates to a method wherein the stealth agent is polyethylene glycol, PEG, or ganglioside.

In a further embodiment of the present specification, the present invention relates to a method in which polyethylene glycol, PEG, is bound to a phospholipid, in particular distearoylphosphatidylethanolamine.

In another embodiment herein, the present invention relates to a method in which a targeting agent is incorporated into a lipid membrane.

In another embodiment of the present specification, the present invention relates to a method wherein the active ingredient is an imaging agent or a therapeutic agent.

In a further embodiment herein, the present invention relates to a method wherein the imaging agent or therapeutic agent is hydrophobic or hydrophilic.

In yet a further embodiment of the present specification, the present specification relates to a method wherein the imaging agent is a dye.

DETAILED DESCRIPTION OF THE INVENTION

The present specification relates to a method for producing liposomes to obtain a high encapsulation efficiency of an encapsulated formulation, excluding the reduced number of production steps, ie, the extrusion steps of conventional liposome production processes. The liposomes of the present invention are intended to deliver therapeutic agents such as anticancer agents, antioxidants, anti-inflammatory agents, antipyretics, antibiotics, antiviral agents, antirheumatic agents, analgesics, growth-factors, or mixtures thereof.

The method herein has the advantage of achieving the same or better efficiency of small molecule encapsulation in targeted liposomes than previous methods without additional processing steps to generate nanoparticles.

In order to increase the encapsulant, a number of conditions were tested at different stages of the production process, with reduced production steps. The test results show that the initial percentage of ethanol has a significant effect on the encapsulation efficiency. A sharp increase in encapsulation efficiency was observed with the ethanol volume higher than 5% conventional (50% compared to the initial aqueous phase) and also with the use of a lower volume of aqueous formulation solution (the sample was then sampled at typical formulation concentration after ethanol evaporation or tangential flow filtration). 5 times diluted to obtain The size of the vesicles was positively affected by the ethanol volume after this ratio was achieved. Indeed, batches with higher ethanol volume (>50%) showed larger vesicles (<150 nm) than previously generated liposome batches. This result could be attributed to the slower diffusion of ethanol associated with the volume increase of the aqueous phase, leading to the formation of larger sized liposomes due to the slow self-assembly of phospholipids. Therefore, since the hydrophilic agent is mainly encapsulated in the liposome aqueous core, the smaller the vesicle size, the smaller the aqueous core volume and the lower the resulting encapsulation efficiency [10, 11]. However, a compromise between encapsulation efficiency and size was obtained using 50% of the initial ethanol volume. Liposomes obtained with this proportion of ethanol exhibited small size (<150 nm) and PDI values (<0.1). Also, under these conditions, the extrusion process should generally be reduced, and a uniform vesicle size is not perfectly necessary.

Liposome production

In one embodiment of the present specification, a liposome composed of DOPE/cholesterol/DSPE-MPEG (54:36:10, molar ratio) was prepared using an ethanol injection method. Briefly, lipids (DOPE, cholesterol, and DSPE-MPEG) were dissolved in ethanol (5% in the conventional ethanol injection method; 50% in the newly presented pre-concentration method, compared to the initial 20% aqueous phase) at 60 °C. .

The solution was injected into an aqueous solution (phosphate buffered saline, PBS) while stirring. This process is carried out at 70° C. and held for the time required to evaporate all the ethanol volume.

In conventional ethanol injection methods, liposomes are extruded to reduce their size. In the pre-concentration method, after ethanol evaporation or tangential flow filtration, the liposome dispersion is diluted 5-fold in PBS (the remaining 80% of the volume is added). Free therapeutics or imaging agents not incorporated into the liposomes were passed through a gel filtration chromatography column (GE Healthcare) with a 5 kDa cut-off (PD-10 desalting column containing 8.3 mL of Sephadex G-25 medium) from the sample. was removed Hydrophilic therapeutics or imaging agents (eg methotrexate and doxorubicin) are present in this aqueous phase in both the existing ethanol injection method and the newly presented pre-concentration method. In remote/active loading, after production in ammonium sulfate (120 mM, pH=8.5), the buffer is changed to Trizma ^® Base sucrose (10%, w/v, buffered at pH 9.0) and empty liposomes are replaced with therapeutic or imaging agents. incubate with

Characterization of liposomes encapsulating methotrexate: a comparative study between different production methods.

Z. Average (d.nm) PDI Encapsulation Efficiency (%) number of steps Pre-concentration method (50% ethanol versus initial aqueous buffer) 103.1 ± 2.333 0.117 ± 0.003 43.4 2 (no extrusion) 66% ethanol 343,3 ± 9,136 0.067 ± 0.029 41,6 2 (no extrusion) 66% ethanol 124,7 ± 0,635 0.061 ± 0.009 8,3 3 (extrusion) 33% ethanol 99.66 ± 0.095 0.240 ± 0.006 7.6 3 (extrusion) Conventional ethanol injection method (5% ethanol) 117.7 ± 1.779 0.053 ± 0.018 5.7 3 (extrusion)

Table II - Characterization of liposomes encapsulating doxorubicin: a comparative study between different production methods. Z. Average (d.nm) PDI Encapsulation Efficiency (%) number of steps Pre-concentration method (50% ethanol versus initial 20% aqueous buffer) 131.2 ± 1.124 0.109 ± 0.010 84.9 2 (no extrusion) Conventional ethanol injection method (5% ethanol) 115.1 ± 0.551 0.048 ± 0.027 85.1 3 (extrusion) Remote/Active Loading 124.8 ± 1.825 0.132 ± 0.020 76.1 5 (extrusion)

Table III - A-Z List of Cancer Drugs Including Combination Therapeutics A L Abemaciclib (Abemaciclib) Lanreotide Acetate Abiraterone Acetate Lapatinib Ditosylate Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation) Lartruvo (Olaratumab) ABVD Lenalidomide ABVE Lenvatinib Mesylate ABVE-PC Lenvima (Lenvatinib Mesylate) AC Letrozole Acalabrutinib (Acalabrutinib) Leucovorin Calcium AC-T Leukeran (Chlorambucil) Actemra (Tocilizumab) Leuprolide Acetate Adcetris (Brentuximab Vedotin) Levulan Kerastik　(Aminolevulinic Acid) ADE Libtayo (Cemiplimab-rwlc) Ado-Trastuzumab Emtansine LipoDox (Doxorubicin Hydrochloride Liposome) Adriamycin (Doxorubicin Hydrochloride) Lomustine Afatinib Dimaleate Lonsurf (Trifluridine and Tipiracil Hydrochloride) Afinitor (Everolimus) Lupron (Leuprolide Acetate) Akynzeo (Netupitant and Palonosetron Hydrochloride) Lupron Depot (Leuprolide Acetate) Aldara (Imiquimod) Lutathera (Lutetium Lu 177-Dotatate) Aldesleukin Lutetium (Lu 177-Dotatate) Alecensa (Alectinib) Lynparza (Olaparib) Alectinib Alemtuzumab M Alimta (Pemetrexed Disodium) Marqibo (Vincristine Sulfate Liposome) Aliqopa (Copanlisib Hydrochloride) Matulane (Procarbazine Hydrochloride) Alkeran for Injection (Melphalan Hydrochloride) Mechlorethamine Hydrochloride (Mechlorethamine Hydrochloride) Alkeran Tablets (Melphalan) Megestrol Acetate Aloxi (Palonosetron Hydrochloride) Mekinist (Trametinib) Alunbrig (Brigatinib) Mektovi (Binimetinib) Ameluz (Aminolevulinic Acid) Melphalan Amipostine Melphalan Hydrochloride Aminolevulinic Acid Mercaptopurine Anastrozole Mesna (Mesna) Apalutamide Mesnex (Mesna) Aprepitant Methotrexate Aranesp (Darbepoetin Alfa) Methylnaltrexone Bromide Aredia (Pamidronate Disodium) Midostaurin Arimidex (Anastrozole) Mitomycin C (mitomycin C) Aromasin (Exemestane) Mitoxantrone Hydrochloride Arranon (Nelarabine) Mogamulizumab-kpkc (mogamulizumab-kpkc) Arsenic Trioxide Mozobil (Plerixafor) Arzerra (Ofatumumab) Mustargen (Mechlorethamine Hydrochloride)
(Mechlorethamine Hydrochloride) Asparaginase Erwinia chrysanthemi MVAC Atezolizumab Myleran (Busulfan) Avastin (Bevacizumab) Mylotarg (Gemtuzumab Ozogamicin) Avelumab Axicabtagene Ciloleucel N Axitinib (Axitinib) Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation) Azacitidine (Azacitidine) Navelbine (Vinorelbine Tartrate) Azedra (Iobenguane I 131) Necitumumab Nelarabine B Neratinib Maleate Bavencio (Avelumab) Nerlynx (Neratinib Maleate) BEACOPP Netupitant and Palonosetron Hydrochloride Beleodaq (Belinostat) Neulasta (Pegfilgrastim) Belinostat Neupogen (Filgrastim) Bendamustine Hydrochloride (Bendamustine Hydrochloride) Nexavar (Sorafenib Tosylate) Bendeka (Bendamustine Hydrochloride) Nilandron (Nilutamide) BEP Nilotinib (Nilotinib) Besponsa (Inotuzumab Ozogamicin) Nilutamide Bevacizumab Ninlaro (Ixazomib Citrate) Bexarotene Niraparib Tosylate Monohydrate Bicalutamide Nivolumab BiCNU (Carmustine) Nplate (Romiplostim) Binimetinib Bleomycin (Bleomycin) O Blinatumomab (blinatumomab) Obinutuzumab Blincyto (Blinatumomab) Odomzo (Sonidegib) Bortezomib OEPA Bosulif (Bosutinib) Ofatumumab Bosutinib (bosutinib) OFF Braftovi (Encorafenib) Olaparib Brentuximab Vedotin Olaratumab Brigatinib (Brigatinib) Omacetaxine Mepesuccinate BuMel Oncaspar (Pegaspargase) Busulfan Ondansetron Hydrochloride Busulfex (Busulfan) Onivyde (Irinotecan Hydrochloride Liposome) Ontak (Denileukin Diftitox) C Opdivo (Nivolumab) Cabazitaxel OPPA Cabometyx (Cabozantinib-S-Malate) Osimertinib (Osimertinib) Cabozantinib-S-Malate Oxaliplatin CAF Calquence (Acalabrutinib) P Campath (Alemtuzumab) Paclitaxel Camptosar (Irinotecan Hydrochloride) Paclitaxel Albumin-stabilized Nanoparticle Formulation Capecitabine PAD CAPOX Palbociclib Carac (Fluorouracil--Topical) (
fluorouracil--topical) Palifemin Carboplatin Palonosetron Hydrochloride CARBOPLATIN-TAXOL Palonosetron Hydrochloride and Netupitant Carfilzomib (carfilzomib) Pamidronate Disodium Carmustine Panitumumab (panitumumab) Carmustine Implant Panobinostat (panobinostat) Casodex (Bicalutamide) Pazopanib Hydrochloride CEM PCV Cemiplimab-rwlc (semiplimab-rwlc) PEB Ceritinib (Ceritinib) Pegaspargase Cerubidine (Daunorubicin Hydrochloride) Pegfilgrastim Cervarix (Recombinant HPV Bivalent Vaccine) Peginterferon Alfa-2b Cetuximab (cetuximab) PEG-Intron (Peginterferon Alfa-2b) CEV Pembrolizumab Chlorambucil Pemetrexed Disodium CHLORAMBUCIL-PREDNISONE (chlorambucil-prednisone) Perjeta (Pertuzumab) CHOP Pertuzumab Cisplatin Plerixafor Cladribine Pomalidomide Clofarabine Pomalyst (Pomalidomide) Clolar (Clofarabine) Ponatinib Hydrochloride CMF Portrazza (Necitumumab) Cobimetinib (Cobimetinib) Poteligeo (Mogamulizumab-kpkc) Cometriq (Cabozantinib-S-Malate) Pralatrexate Copanlisib Hydrochloride (Copanlisib Hydrochloride) Prednisone COPDAC Procarbazine Hydrochloride Copiktra (Duvelisib) Procrit (Epoetin Alfa) COPP Proleukin (Aldesleukin) COPP-ABV Prolia (Denosumab) Cosmegen (Dactinomycin) Promacta (Eltrombopag Olamine) Cotellic (Cobimetinib) Propranolol Hydrochloride Crizotinib (Crizotinib) Provenge (Sipuleucel-T) CVP Purinethol (Mercaptopurine) Cyclophosphamide Purixan (Mercaptopurine) Cyramza (Ramucirumab) Cytarabine (Cytarabine) Q Cytarabine Liposome [No Item] Cytosar-U (Cytarabine) R D Radium 223 Dichloride Dabrafenib (dabrafenib) Raloxifene Hydrochloride (Raloxifene Hydrochloride) Dacarbazine Ramucirumab Dacogen (Decitabine) Rasburicase (Rasburicase) Dacomitinib (dacomitinib) R-CHOP Dactinomycin R-CVP Daratumumab Recombinant Human Papillomavirus (HPV) Bivalent Vaccine Darbepoetin Alfa Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine Darzalex (Daratumumab) Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine Dasatinib (Dasatinib) Recombinant Interferon Alfa-2b Daunorubicin Hydrochloride (Daunorubicin Hydrochloride) Regorafenib Daunorubicin Hydrochloride and Cytarabine Liposome Relistor (Methylnaltrexone Bromide) Decitabine R-EPOCH (R-Epoch) Defibrotide Sodium Retacrit (Epoetin Alfa) Defitelio (Defibrotide Sodium) Revlimid (Lenalidomide) Degarelix Rheumatrex (Methotrexate) Denileukin Diftitox Ribociclib (Ribociclib) Denosumab R-ICE DepoCyt (Cytarabine Liposome) (Cytarabine Liposome) Rituxan (Rituximab) Dexamethasone (dexamethasone) Rituxan Hycela (Rituximab and Hyaluronidase Human) Dexrazoxane Hydrochloride Rituximab (rituximab) Dinutuximab (Dinutuximab) Rituximab and Hyaluronidase Human Docetaxel Rolapitant Hydrochloride Doxil (Doxorubicin Hydrochloride Liposome) Romidepsin Doxorubicin Hydrochloride (Doxorubicin Hydrochloride) Romiplostim Doxorubicin Hydrochloride Liposome Rubidomycin (Daunorubicin Hydrochloride) Dox-SL (Doxorubicin Hydrochloride Liposome) (Dox-SL (Doxorubicin Hydrochloride Liposome)) Rubraca (Rucaparib Camsylate) Durvalumab Rucaparib Camsylate Duvelisib Ruxolitinib Phosphate Rydapt (Midostaurin) E Efudex (Fluorouracil--Topical) S Eligard (Leuprolide Acetate) Sancuso (Granisetron) Elitek (Rasburicase) Sclerosol Intrapleural Aerosol (Talc) Ellence (Epirubicin Hydrochloride) Siltuximab (siltuximab) Elotuzumab Sipuleucel-T (Sipuleucel-T) Eloxatin (Oxaliplatin) Somatuline Depot (Lanreotide Acetate) Eltrombopag Olamine Sonidegib Emend (Aprepitant) Sorafenib Tosylate Empliciti (Elotuzumab) Sprycel (Dasatinib) Enasidenib Mesylate STANFORD V Encorafenib (encorafenib) Sterile Talc Powder (Talc) Enzalutamide Steritalc (Talc) Epirubicin Hydrochloride Stivarga (Regorafenib) EPOCH Sunitinib Malate Epoetin Alfa Sustol (Granisetron) Epogen (Epoetin Alfa) Sutent (Sunitinib Malate) Erbitux (Cetuximab) Sylatron (Peginterferon Alfa-2b) Eribulin Mesylate Sylvant (Siltuximab) Erivedge (Vismodegib) Synribo (Omacetaxine Mepesuccinate) Erleada (Apalutamide) Erlotinib Hydrochloride T Erwinaze (Asparaginase Erwinia chrysanthemi) Tabloid (Thioguanine) Ethyol (Amifostine) TAC Etopophos (Etoposide Phosphate) Tafinlar (Dabrafenib) Etoposide Tagrisso (Osimertinib) Etoposide Phosphate Talc (talc) Evacet (Doxorubicin Hydrochloride Liposome) Talimogene Laherparepvec Everolimus Tamoxifen Citrate Evista (Raloxifene Hydrochloride) Tarabine PFS (Cytarabine) Evomela (Melphalan Hydrochloride) Tarceva (Erlotinib Hydrochloride) Exemestane Targretin (Bexarotene) Tasigna (Nilotinib) F Tavalisse (Fostamatinib Disodium) 5-FU (Fluorouracil Injection) Taxol (Paclitaxel) 5-FU (Fluorouracil--Topical) Taxotere (Docetaxel) Fareston (Toremifene) Tecentriq (Atezolizumab) Farydak (Panobinostat) Temodar (Temozolomide) Faslodex (Fulvestrant) Temozolomide FEC Temsirolimus Femara (Letrozole) Thalidomide Filgrastim Thalomid (Thalidomide) Firmagon (Degarelix) Thioguanine Fludarabine Phosphate Thiotepa Fluoroplex (Fluorouracil--Topical) Tibsovo (Ivosidenib) Fluorouracil Injection Tisagenlecleucel (Tisagenlecleucel) Fluorouracil--Topical Tocilizumab Flutamide Tolak (Fluorouracil--Topical) FOLFIRI Topotecan Hydrochloride FOLFIRI-BEVACIZUMAB (Folfiri-bevacizumab) Toremifene FOLFIRI-CETUXIMAB (Folfiri-cetuximab) Torisel (Temsirolimus) FOLFIRINOX Totect (Dexrazoxane Hydrochloride) FOLFOX TPF Folotyn (Pralatrexate) Trabectedin Fostamatinib Disodium Trametinib (trametinib) FU-LV Trastuzumab Fulvestrant Treanda (Bendamustine Hydrochloride) Fushilev (Leucovorin Calcium) Trexall (Methotrexate) Trifluridine and Tipiracil Hydrochloride G Trisenox (Arsenic Trioxide) Gardasil (Recombinant HPV Quadrivalent Vaccine) Tykerb (Lapatinib Ditosylate) Gardasil 9 (Recombinant HPV Nonavalent Vaccine) Gazyva (Obinutuzumab) U Gefitinib (Gefitinib) Unituxin (Dinutuximab) Gemcitabine Hydrochloride (Gemcitabine Hydrochloride) Uridine Triacetate GEMCITABINE-CISPLATIN (gemcitabine-cisplatin) GEMCITABINE-OXALIPLATIN (gemcitabine-oxaliplatin) V Gemtuzumab Ozogamicin VAC Gemzar (Gemcitabine Hydrochloride) Valrubicin Gilotrif (Afatinib Dimaleate) Valstar (Valrubicin) Gleevec (Imatinib Mesylate) Vandetanib Gliadel Wafer (Carmustine Implant) VAMP Glucarpidase Varubi (Rolapitant Hydrochloride) Goserelin Acetate Vectibix (Panitumumab) Granisetron (granisetron) VeIP Granisetron Hydrochloride (Granisetron Hydrochloride) Velcade (Bortezomib) Granix (Filgrastim) Vemurafenib (Vemurafenib) Venclexta (Venetoclax)
Venetoclax)) H Venetoclax Halaven (Eribulin Mesylate) Verzenio (Abemaciclib) Hemangeol (Propranolol Hydrochloride) Vidaza (Azacitidine) Herceptin (Trastuzumab) Vinblastine Sulfate (Vinblastine Sulfate) HPV Bivalent Vaccine, Recombinant (HPV Bivalent Vaccine, Recombinant) Vincristine Sulfate (Vincristine Sulfate) HPV Nonavalent Vaccine, Recombinant Vincristine Sulfate Liposome HPV Quadrivalent Vaccine, Recombinant (HPV Quadrivalent Vaccine, Recombinant) Vinorelbine Tartrate Hycamtin (Topotecan Hydrochloride) VIP Hydrea (Hydroxyurea) Vismodegib Hydroxyurea Vistogard (Uridine Triacetate) Hyper-CVAD Vizimpro (Dacomitinib) Voraxaze (Glucarpidase) I Vorinostat (Vorinostat) Ibrance (Palbociclib) Votrient (Pazopanib Hydrochloride) Ibritumomab Tiuxetan Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome) Ibrutinib (Ibrutinib) ICE W Iclusig (Ponatinib Hydrochloride) [No Item] Idarubicin Hydrochloride Idelalisib X Idhifa (Enasidenib Mesylate)
(enasidenib mesylate)) Xalkori (Crizotinib) Ifex (Ifosfamide) Xeloda (Capecitabine) Ifosfamide XELIRI (Jelly) IL-2 (Aldesleukin) (IL-2 (Aldesleukin)) XELOX Imatinib Mesylate Xgeva (Denosumab) Imbruvica (Ibrutinib) Xofigo (Radium 223 Dichloride) Imfinzi (Durvalumab) Xtandi (Enzalutamide) Imiquimod Imlygic (Talimogene Laherparepvec) Y Inlyta (Axitinib) Yervoy (Ipilimumab) Inotuzumab Ozogamicin Yescarta (Axicabtagene Ciloleucel) Interferon Alfa-2b, Recombinant Yondelis (Trabectedin) Interleukin-2 (Aldesleukin) Intron A (Recombinant Interferon Alfa-2b) Z Iobenguane I 131 Zaltrap (Ziv-Aflibercept) Ipilimumab Zarxio (Filgrastim) Iressa (Gefitinib) Zejula (Niraparib Tosylate Monohydrate) Irinotecan Hydrochloride Zelboraf (Vemurafenib) Irinotecan Hydrochloride Liposome Zevalin (Ibritumomab Tiuxetan) Istodax (Romidepsin) Zinecard (Dexrazoxane Hydrochloride) Ivosidenib (Ivosidenib) Ziv-Aflibercept (Ziv-Aflibercept) Ixabepilone Zofran (Ondansetron Hydrochloride) Ixazomib Citrate Zoladex (Goserelin Acetate) Ixempra (Ixabepilone) Zoledronic Acid Zolinza (Vorinostat) J Zometa (Zoledronic Acid) Jakafi (Ruxolitinib Phosphate) Zydelig (Idelalisib) JEB Zykadia (Ceritinib) Jevtana (Cabazitaxel) Zytiga (Abiraterone Acetate) K Kadcyla (Ado-Trastuzumab Emtansine) Kepivance (Palifermin) Keytruda (Pembrolizumab) Kisqali (Ribociclib) Kymriah (Tisagenlecleucel) Kyprolis (Carfilzomib)

Table IV- A to Z list of drugs used to treat rheumatoid arthritis A M Abaloparatide (parathyroid hormone) Magnacet Abatacept Maxidone Acetaminophen (children and infants) Meclofenamate sodium Acetaminophen 325mg (Acetaminophen 325mg) Mediproxen Acetaminophen 500 mg (Acetaminophen 500 mg) Medrol Acetaminophen 650 mg (Acetaminophen 650 mg) Mefenamic acid Acetaminophen with codeine Meloxicam Acetylsalicylic acid (aspirin) Menest Actemra Menostar Activella Methadone hydrochloride (methadone hydrochloride) Actonel Methadose (Methadose) Adalimumab Methotrexate Addaprin Methylprednisolone Advil Miacalcin Alendronate Millipred Alendronate with vitamin D Milnacipran Aleve Mitigare Allopurinol Mobic Ambien Morphine sulfate Ambien CR Morphine sulfate oral solution Amitriptyline hydrochloride Morphine sulfate with naltrexone Amrix Motrin Anacin Motrin IB Anacin (aspirin free) MS Contin Anakinra Mycophenolate mofetil Anaprox Anaprox DS N Apremilast Nabumetone Arava Nalfon Arthrotec Naprelan Atelvia Naprosyn Avinza Naproxen and esomeprazole magnesium Azasan Naproxen sodium (over-the-counter) Azathioprine Naproxen sodium (prescription) Azulfidine Neoral Azulfidine EN-Tabs Neurontin Norco B Baricitinib (baricitinib) O Baycadron Olumiant Bayer Opana Belimumab Oramorph SR Benlysta Orapred Betamethasone Orencia Binosto Otezla Boniva Otrexup Brisdelle Oxaprozin Bufferin Oxycodone Oxycodone hydrochloride with acetaminophen C Oxycodone with aspirin Calcitonin (nasal spray) Oxycodone with ibuprofen Cambia OxyContin Canakinumab Oxymorphone hydrochloride Cataflam Celebrex P Celecoxib (celecoxib) Paroxetine Celeston Paxil CellCept PediaCare Fever Reducer/Pain Reliever Cequa (solution) Pediapred Certolizumab pegol Pegloticase Cevimeline Pennsaid Children's Tylenol Percocet Cimzia Percodan Climara Pro Pexeva Clinoril Pilocarpine Cocet Piroxicam Cocet Plus Plaquenil Colchicine Ponstel Colcrys Prednisolone Combunox Prednisone Conjugated estrogens/bazedoxifene Prednisone Intensol ConZip Pregabalin Cortef Prelone Cortisone acetate Premphase Cosentyx Prempro Cyclobenzaprine (Cyclobenzaprine) Primlev Cyclophosphamide Probenecid Cyclosporine Probenecid and colchicine Cyclosporine ophthalmic emulsion/solution Prolia Cymbalta Prozac D R Daypro Raloxifene hydrochloride Denosumab Rasuvo Dexamethasone (dexamethasone) Rayos (Rayos) DexPak Reclast Diclofenac Remicade Diclofenac potassium Renflexis (infliximab-abda, biosimilar to Remicade) Diclofenac sodium Reprexain Diclofenac Sodium liquid/gel Restasis (emulsion) Diclofenac sodium with misoprostol Rheumatrex Diflunisal Risedronate sodium Dolophine Rituxan (Rituxan) Duavee Rituximab (rituximab) Duexis Roxicet Duloxetine Roxicodone Dyspel Rybix ODT Ryzolt E EC-Naprosyn (EC-Naprosyn) S Ecotrin Salagen Effexor XR Sandimmune Embeda Sarafem Enbrel Sarilumab Endocet Savella Endodan secukinumab Estrace Sertraline Estratab Simponi, Simponi Aria Estrogens Stellara Estrogens with progesterone Sulfasalazine Etanercept Sulfazine Etodolac Sulfazine EC Evista Sulindac Evoxac Excedrin T Taltz F Teriparatide (parathyroid hormone) Febuxostat TH Ibuprofen (TH Ibuprofen) Feldene Therafeldamine Fenoprofen calcium Tivorbex FeverAll Tocilizumab Fexmid Tofacitinib (extended release) Flexeril Tofacitinib (immediate release) Fluoxetine Tolmetin sodium Flurbiprofen (flurbiprofen) Tramadol Forteo Tramadol (extended release) Fortical Tramadol with acetaminophen Fosamax Trexall Fosamax Plus D Tylenol Arthritis Pain Tylenol Extra Strength G Tylenol Regular Strength Gabapentin Tylenol with Codeine No.2 Gengraf Tylenol with Codeine No.3 Genpril Tylenol with Codeine No.4 Golimumab Tymlos Gralis U H Uloric Horizant Ultracet Humira Ultram Hycet Ultram-ER Hydrocet Ustekinumab Hydrocodone bitartrate Hydrocodone bitartrate with acetaminophen V Hydrocodone bitartrate with ibuprofen Venlafaxine Hydrocortisone Veripred 20 Hydrogesic Vicodin Hydroxychloroquine sulfate Vicoprofen Hydroxypropyl cellulose pellets Vimovo Hysingla ER Vivlodex Voltaren I Voltaren XR Ibandronate Ibuprofen (over-the-counter) X Ibuprofen (prescription) Xatmep Ibuprofen with famotidine Xeljanz (Xeljanz) Ilaris Xeljanz XR Imuran Xodol Indocin Xolox Indomethacin Infant's Tylenol Z Inflectra (infliximab-dyyb, biosimilar to Remicade) Zamicet Infliximab (infliximab) Zipsor Intermezzo Zohydro ER I-Prin Zoledronic acid ixekizumab Zoloft Zolpidem K Zolvit Kadian Zometa Ketoprofen Zorvolex Kevzara Zurampic (Zurampic) Kineret Zydone Krystexxa (Krystex) Zyloprim KS Ibuprofen (KS Ibuprofen) L Lacrisert Leflunomide lesinurad Lorcet Lortab Lyrica

Table IV List of Dyes A to Z

In this specification, where ranges are provided, endpoints are included. Further, unless otherwise indicated or otherwise apparent from context and/or understanding of one of ordinary skill in the art, values expressed as ranges, unless the context clearly dictates otherwise, include up to tenths of the unit of the lower limit of the range. It should be understood that the embodiments may take any specific value within the recited ranges. Unless otherwise indicated or otherwise apparent from the context and/or understanding of one of ordinary skill in the art, a value expressed in a range may take any sub-range within a given range, wherein the endpoint of the sub-range is one tenth of the unit of the lower limit of the range. expressed with the same accuracy as

The term "comprising" whenever used herein is intended to indicate the presence of a stated feature, integer, step, element, but of one or more other features, integers, steps, elements, or groups thereof. It does not exclude the presence or addition.

Of course, this specification is not in any way limited to the described embodiments, and those skilled in the art will anticipate many possibilities for modifications thereof without departing from the basic spirit of the specification as defined in the appended claims.

The above-described embodiments are obviously combinable.

The following dependent claims set forth specific embodiments of the invention.

sequence list

SEQ-ID. NO.1: Folic acid-DRDDQAAWFSQY

SEQ-ID. NO 2: KDEPQRRRSARLSAKPAPPKPEPKPKKAPAKK-DRDDQAAWFSQY

SEQ-ID. NO 3: YQSFWAAQDDRD-KDEPQRRRSARLSAKPAPPKPEPKPKKAPAKK

References

[One] Wang, A. Z., Langer, R., and Farokhzad, O. C., Nanoparticle Delivery of Cancer Drugs, Annual Review of Medicine. 2012, 63, 185-198.

[2] Hong, M.-S., Lim, S.-J., Lee, M.-K., Kim, YB, and Kim, C.-K., Prolonged Blood Circulation of Methotrexate by Modulation of Liposomal Composition, Drug Delivery . 2001, 8, 231-237.

[3] Laouini, A., Jaafar-Maalej, C., Limayem-Blouza, I., Sfar, S., Charcosset, C., and Fessi, H., Preparation, Characterization and Applications of Liposomes: State of the Art, Journal of Colloid Science and Biotechnology. 2012, 1, 147-168.

[4] Bangham, A. D., Standish, M. M., and Watkins, J. C., Diffusion of univalent ions across the lamellae of swollen phospholipids, Journal of Molecular Biology. 1965, 13, 238-IN27.

[5] Batzri, S. and Korn, E. D., Single bilayer liposomes prepared without sonication, Biochimica et Biophysica Acta (BBA) - Biomembranes. 1973, 298, 1015-1019.

[6] Naeff, R., Feasibility of topical liposome drugs produced on an industrial scale, Advanced Drug Delivery Reviews. 1996, 18, 343-347.

[7] Sur, S., Fries, A. C., Kinzler, K. W., Zhou, S., and Vogelstein, B., Remote loading of preencapsulated drugs into stealth liposomes, Proceedings of the National Academy of Sciences of the United States of America. 2014, 111, 2283-2288.

[8] Gubernator, J., Active methods of drug loading into liposomes: Recent strategies for stable drug entrapment and increased in vivo activity. Vol. 8. 2011. 565-80.

[9] Duong, A. D., Collier, M. A., Bachelder, E. M., Wyslouzil, B. E., and Ainslie, K. M., One Step Encapsulation of Small Molecule Drugs in Liposomes via Electrospray-Remote Loading, Mol Pharm. 2016, 13, 92-9.

[10] Jaafar-Maalej, C., Diab, R., Andrieu, V., Elaissari, A., and Fessi, H., Ethanol injection method for hydrophilic and lipophilic drug-loaded liposome preparation, J Liposome Res. 2010, 20, 228-43.

[11] Pons, M., Foradada, M., and Estelrich, J., Liposomes obtained by the ethanol injection method, International Journal of Pharmaceutics. 1993, 95, 51-56.

SEQUENCE LISTING <110> UNIVERSIDADE DO MINHO <120> METHOD FOR PRODUCTION OF LIPOSOMES <130> P687.3 WO <150> PT115500 <151> 2019-05-07 <160> 3 <170> BiSSAP 1.3.6 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Folic Acid with SEQ ID 1 <400> 1 Asp Arg Asp Asp Gln Ala Ala Trp Phe Ser Gln Tyr 1 5 10 <210> 2 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Sequence 2 <400> 2 Lys Asp Glu Pro Gln Arg Arg Ser Ala Arg Leu Ser Ala Lys Pro Ala 1 5 10 15 Pro Pro Lys Pro Glu Pro Lys Pro Lys Lys Ala Pro Ala Lys Lys Asp 20 25 30 Arg Asp Asp Gln Ala Ala Trp Phe Ser Gln Tyr 35 40 <210> 3 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Sequence 3 <400> 3 Tyr Gln Ser Phe Trp Ala Ala Gln Asp Asp Arg Asp Lys Asp Glu Pro 1 5 10 15 Gln Arg Arg Ser Ala Arg Leu Ser Ala Lys Pro Ala Pro Pro Lys Pro 20 25 30 Glu Pro Lys Pro Lys Lys Ala Pro Ala Lys Lys 35 40

Claims (23)

preparing an ethanolic phase by mixing phospholipids and hydrophobic molecules of a steroid with ethanol;
preparing an aqueous phase with the active ingredient and the targeting agent in a buffer solution;
obtaining liposomes by injecting the ethanol phase into the aqueous phase at a temperature of about 50° C. to about 80° C., wherein the ethanol/aqueous phase volume ratio is 1:1 to 3:2;
remove ethanol; and
removing the remaining free active ingredient in an appropriate manner, ie by tangential flow filtration;
A method for encapsulating an active ingredient in liposomes comprising sequential steps, comprising:
The method of claim 1, wherein the targeting agent is a peptide conjugated to a liposome component or incorporated into a lipid membrane.
The method of claim 1 , wherein the ethanol removal is by evaporation or tangential flow filtration.
3. The method according to claim 1 or 2, wherein the steroid is cholesterol.
4. The method according to any one of claims 1 to 3, wherein the cholesterol is cholesteryl hemisuccinate.
5. The method according to any one of claims 1 to 4, further comprising diluting the liposome dispersion 1 to 10 fold in the further diluted aqueous phase.
6 . The method of claim 1 , wherein the ethanol phase is injected at a rate of approximately 2-4 ml/min.
Process according to any one of the preceding claims, characterized in that the ethanol concentration to the initial aqueous volume is between 40% and 60%, preferably 50%.
Method according to any one of the preceding claims, characterized in that the temperature is 60°C or 70°C.
9. The method of any one of claims 1 to 8, wherein the free active ingredient is a multicharged molecule containing at least one negative charge at a pH of about 4 to about 7.
10. The drug according to any one of claims 1 to 9, wherein the free active ingredient is a drug, in particular an anticancer agent, an antirheumatic agent, an anti-neurodegenous disease drug, an antioxidant drug, an anti-inflammatory agent, an antipyretic agent, an antibiotic drug, an antiviral agent. A method, characterized in that it is a drug, an analgesic, or a combination thereof.
11. The method according to any one of the preceding claims, characterized in that the injecting step is carried out under agitation.
12. The method of any one of claims 1-11, wherein the targeting agent is SEQ- ID. NO 1, SEQ-ID. NO 2 , SEQ- ID. at least one peptide selected from the list having a degree of identity of at least 90% of the sequences of NO 3 , or mixtures thereof.
13. The method according to any one of claims 1 to 12, wherein the aqueous phase is phosphate buffered saline (PBS).
14. The method of any one of claims 1-13, wherein the ethanol phase comprises anionic, neutral, or cationic phospholipids.
15. The method according to any one of claims 1 to 14, wherein the phospholipids in the ethanol phase are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and/or derivatives thereof or mixtures thereof, in particular 1,2-dioleoyl -sn-glycero-3-phosphoethanolamine.
16. The method of any one of claims 1-15, wherein the ethanol phase further comprises a stealth agent, a targeting agent, or a mixture thereof.
17. The method according to any one of claims 1 to 16, wherein the stealth agent is polyethylene glycol, PEG, or ganglioside.
18. The method according to any one of the preceding claims, characterized in that the polyethylene glycol (PEG) is bound to a phospholipid, in particular to distearoylphosphatidylethanolamine.
19. The method of any one of claims 1-18, wherein the targeting agent is incorporated into the lipid membrane.
20. A method according to any one of the preceding claims, characterized in that the free active ingredient is an imaging agent or a therapeutic agent.
21. The method of any one of claims 1-20, wherein the imaging or therapeutic agent is hydrophobic or hydrophilic.
22. The method according to any one of the preceding claims, wherein the imaging agent is a dye.
23. The method according to any one of the preceding claims, characterized in that the free active ingredient is methotrexate, doxorubicin, or a mixture thereof.